This study examined fifty-four samples of Tribolodon hakonensis, collected from 43 river basins in Japan, for genetic relationships based on DNA sequences of a part (709bp) in the mitochondrial cytochrome b region. A total of 35 haplotypes were identified. The average genetic distance was found to be highest in the Tokai-Hokuriku-Kinki region and lowest in the Chugoku and Kyushu regions. Haplotype diversity was highest in the Hokkaido region and lowest in the Chugoku region. In the Shikoku region, all haplotypes were recorded only in this region. Three groups were recognized in the network: Hokkaido, Tohoku, Kanto-Koshinetsu, and Tokai-Hokuriku-Kinki; Tohoku and Kanto-Koshinetsu; and Tokai-Hokuriku-Kinki, Chugoku, Shikoku, and Kyushu. In addition, two large clades were revealed in the genetic tree with a border line connecting the Wakasa Bay in the Sea of Japan side and the Ise Bay on the Pacific Ocean side, although some haplotypes were distributed across the Itoigawa-Shizuoka tectonic line. Furthermore, deeply differentiated lineages were observed in the upper reaches of large river basins, such as the Kitakami, Kumano, and Niyodo River basins.